RNA interference is the process of sequence-specific post transcriptional gene silencing in cells initiated by double-stranded RNA (dsRNA) that is homologous in sequence to a portion of a targeted mRNA. Introduction of dsRNA into cells leads to the destruction of the endogenous RNAs that share the same sequence as the dsRNA. The dsRNA molecules are cleaved by an RNase III family nuclease called Dicer into short-interfering RNAs (siRNA), which are 19-23 nucleotides (nt) in length. The siRNAs are incorporated into a multicomponent nuclease complex (RISC, RNA-induced silencing complex), which identifies mRNA substrates through their homology to the siRNA, binds to and destroys the targeted mRNA. In mammalian cells, dsRNAs longer than 30 base pairs can activate the dsRNA-dependent kinase PKR and 2′-5′-oligoadenylate synthetase, normally induced by interferon. By virtue of its small size, synthetic siRNA avoids activation of the interferon response. The activated PKR inhibits general translation by phosphorylation of the translation factor eukaryotic initiation factor 2α (eIF2α), while 2′-5′-oligoadenylate synthetase causes nonspecific mRNA degradation via activation of RNase L.
In contrast to the nonspecific effect of long dsRNA, siRNA can mediate selective gene silencing in the mammalian system Hairpin RNA with a short loop and 19 to 27 base pairs in the stem also selectively silences expression of genes that are homologous to the sequence in the double-stranded stem. Mammalian cells can convert short hairpin RNA into siRNA to mediate selective gene silencing.
RISC mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
Studies have shown that 21 nucleotide siRNA duplexes are most active when containing two nucleotide 3′-overhangs. Furthermore, complete substitution of one or both siRNA strands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereas substitution of the 3′-terminal siRNA overhang nucleotides with deoxy nucleotides (2′-H) was shown to be tolerated.
Studies have shown that replacing the 3′-overhanging segments of a 21-mer siRNA duplex having 2 nucleotide 3′ overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to 4 nucleotides on each end of the siRNA with deoxyribonucleotides has been reported to be well tolerated whereas complete substitution with deoxyribonucleotides results in no RNAi activity.
RNA interference is emerging as a promising means for reducing the expression of specific gene products, and thus may be useful for developing therapeutic drugs to treat viral infections, cancers, autoimmune diseases, and other diseases and conditions amenable to treatment by down-regulation of mRNA expression. However, there remains an important need in the art for additional tools and methods to design, produce, formulate, deliver, and use siRNAs as therapeutic tools, including for therapies targeted to specific tissues and cells.